This revised proposal focuses on the genetic and cellular events leading to pulmonary smooth muscle proliferation in lymphangiomyomatosis (LAM), a progressive and often-fatal lung disease affecting almost exclusively women. LAM can occur in women with tuberous sclerosis complex (TSC) or in women who have no signs of TSC (sporadic LAM). Renal angiomyolipomas occur in most TSC patients and in 50% of sporadic LAM patients. In work funded by this grant, we found TSC2 mutations in angiomyolipomas and pulmonary LAM cells from women with sporadic LAM, but not in normal kidney or lung, which strongly suggests that LAM and angiomyolipoma cells are derived from the same cell. This raises the possibility of an unusual disease mechanism for sporadic LAM: the migration of histologically benign smooth muscle cells between angiomyolipoma and lung. Recent work by another group has demonstrated that hamartin (the TSC1 gene product) interacts with the moesin-ezrin-radixin family of cytoskeletal proteins, activates the small GTPase Rho, and regulates cellular adhesion. Our preliminary data indicate that tuberin (the TSC2 gene product) also activates Rho and regulates cell adhesion and migration. Therefore, the central hypothesis of this proposal is that mutational inactivation of either TSC1 or TSC2 leads to LAM because of aberrant signaling in cellular pathways including Rho. To address this hypothesis, we propose the following specific aims: Aim 1: To define the role of TSC1 and TSC2 mutations in LAM. Aim 2: To determine whether LAM cells and angiomyolipoma cells have identical clonality patterns. Aim 3: To determine the mechanisms through which TSC1 and TSC2 affect cell migration. Our long-term goal is to define the cellular pathways through which mutations in the TSC1 and TSC2 genes lead to smooth muscle cell proliferation and LAM. A natural corollary of this goal is to test the hypothesis that LAM and angiomyolipoma cells are derived from the same precursor cell. We are optimistic that this project will contribute to the development of targeted therapeutic strategies for LAM and TSC patients, for whom there are currently limited treatment options. Hamartin and tuberin are expressed in most human tissues, and the manifestations of TSC include tumors of the brain, heart, and kidney, as well as mental retardation, seizures, and autism. We strongly believe, therefore, that the pathways in which hamartin and tuberin participate have broad medical and biological significance.